Knockout mice have proven invaluable in validating new radiotracers as ligands for a specific receptor. The biodistribution of the proposed binding site-specific radioligand is determined in groups of wild-type and knockout mice homozygous (-/-) for the target of interest. The difference in binding of the radioligand in wild-type and knockout mice can be attributed to the absence of the specific binding site, all other variables being equal. This is important given that doses of drug to inhibit binding at the binding site of interest are rarely specific, especially at the doses needed to saturate the binding site. Therefore, knockout mice represent a solution to the time-consuming process of validating new radiotracers that is quantitative and requires minimal experimentation. The early detection of Alzheimer's disease using PET imaging has thus far not been possible, although several radiotracers are being evaluated. The M1 and M2 receptor subtypes might be suitable targets for investigating Alzheimer's disease, although the autopsy studies were small in number and carried out on patients who had been treated with various drugs. Agonists measure both the receptor density and the affinity state of the receptor, whereas antagonists measure only receptor density. Therefore, agonists are likely to be more useful radiotracers {e.g. the M2 receptor agonist [18F]FPTZTP). Based on experiments involving the radiolabeling and validating of [18F]FPTZTP and [18F]paclitaxel (FPAC), the knockout mouse appears to be the most expeditious method for radiotracer validation. The pharmacological approach is time-consuming, and might not give a definitive answer. A smaller number of experiments are involved in radiotracer validation using knockout mice compared with validating a new tracer by testing saturability, specificity and distribution characteristics. The knockout mouse approach is therefore more suitable for drug development. Knockout mice represent a clearly defined biochemical change whereas pharmacological intervention rarely represents a simple biochemical change. Many binding site-specific molecules are not specific at the doses needed to block the binding site in order to prove saturable binding. Regional brain localization of [18F]FPTZTP in M2 receptor knockout mice compared with wild-type mice, M1 receptor knockout mice, M3 receptor knockout mice and M4 receptor knockout mice clearly shows the preference of [18F]FPTZTP for the M2 receptor subtype. With the availability of knockout mice, these validation experiments can be completed in a matter of weeks rather than the months necessary for the full pharmacological approach. Knockout mice have been used to show that [18F]FDG is a sensitive probe of changes in 6-glucose phosphatase (G6Pase) levels. The monitoring of gene therapy of glycogen storage disease type 1a in a mouse model was achieved using [18F]FDG and a dedicated animal scanner. The G6Pase knockout mice were compared with knockout mice infused with a recombinant adenovirus containing the murine gene encoding G6Pase (Ad-mG6Pase). Serial images of the same mouse before and after therapy were obtained and compared with wild-type mice of the same strain to determine the uptake and retention of [18F]FDG in the liver. Image data were acquired from heart, blood and liver 20 min after injection of [18F]FDG. The retention of [18F]FDG was lower for the wild-type mice compared with the knockout mice. The mice treated with adenovirus-mediated gene therapy showed [18F]FDG retention similar to that found in age-matched wild-type mice. These studies show that FDG can be used to monitor G6Pase concentration and, therefore, the progress of glycogen storage disease. [18F]FPAC is a sensitive probe for P-glycoprotein (P-gp), a protein responsible for multidrug resistance. Paclitaxel (Taxol) is a clinically important chemotherapeutic agent. [18F]FPAC shows high uptake into and rapid clearance from tissues in rats. Pre-administration of paclitaxel in rats significantly increases the retention of [18F]FPAC in blood (33.0% increase in retention), heart (32.0%), lung (37.6%) and kidney (142.4%). Biodistribution and radiation dose estimates for [18F]FPAC have been obtained in monkeys, and the effects of a P-gp blocker, XR9576 (Xenova, http://www.xenova.co.uk), on FPAC kinetics have also been studied. Liver uptake of FPAC was significantly affected by XR9576. Studies with mdr1a/1b (-/-) knockout mice showed significant increases in the uptake of [18F]FPAC in the heart, lungs, femur, muscle and brain compared with wild-type mice. Changes in the uptake of [18F]FPAC resulting from pre-injection of unlabeled paclitaxel were significant only in the lung and kidney of wild-type mice. Therefore, [18F]FPAC is a substrate for P-gp and might be useful for in vivo imaging of P-gp-mediated efflux. To apply the information obtained from knockout mice to clinical studies, experiments must be carried out to confirm that the PK and PD are similar. Metabolic differences are the most likely confounding factor. With the availability of mouse and human hepatocytes and the improved sensitivity of LC/MS, metabolite identification in both species can be easily ascertained. The use of LC/MS and hepatocyte preparations allows the differences in metabolism between species to be assessed. In humans, the major use of PET has been, and will continue to be, in occupancy studies, either at a single time point or as a function of time after drug dosing. Driven by many advances in technology and the use of knockout mice and LC/MS, PET imaging is rapidly becoming a major force in drug discovery.